Methylmercury (MeHg) is a prominent environmental neurotoxicant that induces cerebellar toxicity including degeneration of granule neurons. We hypothesize that exposure to MeHg induces disruption of the ability of the cell to regulate intracellular Ca2+ ([Ca2+]i) leading to a cascade of effects including disruption of membrane excitability, impaired synaptic function and ultimately- cell death. During the last funding period we demonstrated unequivocally that MeHg disrupts Ca2+ regulation in granule cells, and does so at submicromolar concentrations. Prevention of Ca2+ entry precluded or delayed MeHg- induced cell death and elevation of [Ca2+]i. Preliminary results now suggest that low concentrations of MeHg cause a more disruptive change in [Ca2+]i in granule cells in culture than in Purkinje cells. Proposed studies in this renewal application aim to follow-up on this observation and will continue to focus on the role of MeHg-induced changes in [Ca2+]i to dysfunction and death of cerebellar granule cells. In particular, comparison will be made of the differences in [Ca2+]i regulation in response to MeHg between granule neurons, and the apparently less sensitive Purkinje neurons. Effects on ion channels, synaptic function and regulation of [Ca2+]i will be examined. Studies will utilize rat cerebellar slices prepared freshly, or used in organotypic culture, granule and Purkinje neurons in culture, mitochondria or microsomes isolated from cerebellar gray matter, or cloned Ca2+ channels expressed in human embryonic kidney (HEK293) cells. Effects of acute and subacute exposure to MeHg will be examined using a combination of molecular, electrophysiological, biochemical, and digital imaging determinations of fluorescent indicator dyes of [Ca2+]i. The three Specific Aims are: 1). Does exposure to MeHg alter the function of specific populations of Ca2+ channels? Are granule and Purkinje cells affected differentially, and are the effects on the channel pore, or on gating/modulating aspects of channel function? 2). Does MeHg disrupt intracellular Ca2+ homeostasis by specific effects on Ca2+ buffering systems? If so, is the effect on the permeability pathways or a secondary result on energy metabolism? Are differences in granule/Purkinje cell sensitivity to MeHg due to differences in their regulation of [Ca2+]i? 3). By what mechanisms does MeHg affect cerebellar synaptic transmission? What is the relationship of the changes in [Ca2+]i induced by MeHg with the disruption of cerebellar synaptic transmission? Does chronic exposure to MeHg cause sustained impairments in cerebellar synaptic function? Results of these studies should lead to better understanding of a) potential sites perturbed after exposure to MeHg, particularly the relationship of early acute actions on membrane and [Ca2+]i handling to the effects on synaptic function neuronal death, and b) explanations for heightened sensitivity of cerebellar granule neurons to MeHg.